Lipid Productivity and Biosynthesis Genes Response of Indigenous Chlorella sp. T4 Strain under Different Nitrogen and Phosphorus Load

Microalgae can synthesize and accumulate high neutral lipids upon exposure to abiotic stress such as nutrient starvation or limitation. In this study, indigenous microalgae Chlorella sp. T4 was cultivated in nitrogen and phosphorus under both limiting and replete conditions. Growth, lipid yield, fatty acid proles and biosynthetic gene expression levels were determined to ascertain cell’s response under these conditions. An impaired cell growth was observed under nitrogen limiting condition, evident by the lowest biomass yield (0.58±0.03 g L −1 ) as revealed by low quantum eciency of photosystem II (Fv/Fm) value and chlorophyll a content. An increase in lipid content yield was observed under nitrogen and phosphorus limiting conditions as compared to the control. Nutrient limiting conditions produced fatty acid methyl ester that is suitable for biodiesel production compared to the control (BG-11). Gene expression analysis using real time q-PCR for photosynthesis (rbcL) and lipid biosynthesis (accD, KAS-1, ω-6 FAD, ω-3 FAD) genes revealed different expression levels under both limiting and replete conditions. Under nutrient limiting conditions, increase in the expression of accD, KAS-1, ω-6 FAD and ω-3 FAD genes was observed, whereas a decrease in rbcL gene expression level was noted. A signicant correlation could be drawn between the expression levels of the biosynthetic genes and growth rate, biomass yield, physiological response, lipid yield and fatty acid composition. These results provide an insight into the physiological response and gene expression level under different nutrient levels, which could be harnessed for future genetic engineering of Chlorella sp. T4 for improved lipid production.


Introduction
The demand for biodiesel, as a replacement for the conventional fossil fuel, is growing worldwide in recent years [1]. Microalgae have been a favourable feedstock for biodiesel production due to its fast growth rate, high lipid yield, suitable fatty acid composition and adaptability to a wide range of climatic environment [2,3], but high production cost at commercial scale is still a major drawback [4]. Microalgae tend to accumulate energy storage material in form of lipids and starch under stress conditions, when the cell growth reaches the stationary phase [5]. It has been shown that optimization of microalgae culture condition can result into high lipid productivities in microalgal biomass. Manipulation of several key intrinsic and extrinsic factors such as nutrient stress, light intensity, temperature, and carbon source triggers lipid accumulation pathway [6][7][8]. Nutrient deprivation is often used by many researchers to improve overall microalgal lipid productivity, due to it's low cost and easy application during the cultivation process [9]. This approach causes decrease in photosynthetic rates, and compromises biomass accumulation, while resulting in enhanced overall lipid storage in form of triacylglycerol [10,11].
Nitrogen (N) is regarded as important micro-nutrient for microalgal growth, as it is associated with protein synthesis and cell division [11], whereas phosphorus (P) contributes to various metabolic processes such as signaling pathways, energy generation and photosynthesis.
Most studies have been focused on obtaining high lipid productivity yield under nutrient-stress conditions, without a proper understanding of the effect of these conditions on the microalgae photosynthesis activity, physiological response, and gene expression levels under these conditions [12,13]. The understanding of microalgal response at the molecular level is limiting to few species, such as Chlamydomonas reinhardtti, Thalassiosira psuedonana and Dunaliella salina [14]. The microalgae under study, Chlorella sp. T4 strain, isolated in our laboratory, have demonstrated huge potential to accumulate large amount of fatty acid that can be used for biodiesel production [15]. In this study, different concentrations of N and P were applied to trigger hyper lipid accumulation in this indigenous microalga. Furthermore, the physiological response of the microalgae under different nutrient conditions and expression levels of ve key fatty acid biosynthetic genes (rbcL, accD, KAS-1, ω-6 FAD and ω-3 FAD) were investigated. The rbcL gene encodes the catalytic large subunit of the enzyme RuBisCO (ribulose 1.5bisphosphate carboxylase/oxygenase) which is responsible for carbon xation, catalyzing the rst step in the Calvin cycle [14]. Previous study reported a decrease in rbcL gene expression under N and P de cient conditions in Chlorella sorokiniana [3]. accD encodes for acetyl-coenzyme A carboxylase carboxyl transferase subunit beta which is responsible for fatty acid biosynthesis, and catalyses the conversion of acetyl-CoA to malonyl-CoA during the lipid biosynthesis [6]. An increase in accD expression under nutrient de cient conditions has been reported in Chlorella pyrenoidosa [16]. The expression levels of three fatty acid biosynthetic genes; KAS-1, ω-6 FAD and ω-3 FAD in relation to fatty acid yield under different N and P concentrations have been investigated. The KAS-1 gene encodes for ketoacyl-ACP synthase-1 responsible for the addition of malonyl-CoA to elongate 4-carbon fatty acid to 6-, 12-and 16 carbon fatty acid chains, for the production of palmitic and stearic acid. Furthermore, ω-6 FAD gene which encodes for omega-6 desaturase responsible for catalyzing the conversion of oleic acid into linoleic acid, while ω-3 FAD gene code for omega-3 desaturase which is responsible for the conversion of ω-6 fatty acid into ω-3 fatty acid [17][18][19]. Therefore, the aim of the present study was to design suitable cultivation conditions for high lipid accumulation that can be used for biodiesel production. To understand the expression of key functional genes (rbcL, accD, KAS-1, ω-6 FAD and ω-3 FAD) by varying N and P concentration in the growth medium.

Algal strain and seed preparation
The microalgal strain Chlorella sp. T4 used in this study was isolated from freshwater body in KwaZulu-Natal, South Africa [15]. The strain was preserved in BG-11 medium which is composed of (g L −1 ): The culture (10% v/v) was inoculated in 500 mL conical Erlenmeyer aks containing 200 mL of BG-11 medium. An aliquot of tetracycline (0.5 µl mL −1 ) was added to the growth medium to prevent any bacterial contamination in the microalgal samples. The culture was incubated at 25°C under cool white uorescent illumination of 200 µmol m −2 s −1 with a photoperiod of 12h: 12h, light: dark cycle under ambient CO 2 . Similar cultivation conditions were maintained for all the subsequent experiments. The cultures were hand-shaken two to three times daily to avoid settling and sticking of the culture onto the bottom of the ask.

Experimental design and physiological parameters analysis
To nd the best nitrogen (N) and phosphorus (P) concentration for high lipid productivity yield, Chlorella sp. T4 was cultivated in BG-11 medium containing sodium nitrates (0.75 and 2.25 g L −1 ) and dipotassium-ortho-phosphate (0.02 and 0.06 g L −1 ). These nutrient concentrations were selected based on the lipid productivity yield obtained from the preliminary study conducted by growing the microalgae strain on BG-11 containing different concentrations of N (0, 0.35, 0.75, 2.25 g L −1 ) and P (0, 0.02, 0.04, 0.06 g L −1 ). Optimization was conducted with one factor at a time, and other individual media composition kept constant as in BG-11 to assess the individual effect of the culture treatment on Chlorella sp. T4. In addition, control experiment was conducted using BG-11 medium with normal concentration of sodium nitrates (1.5 g L −1 ) and di-potassium-ortho-phosphate (0.04 g L −1 ) that is known to support microalgae growth. The algal cell was standardized to the optical density of 0.05 at 680 nm.
The cells were harvested by centrifugation at 5000 rpm for 10 min, washed with distilled water, and resuspended into appropriate medium containing different concentrations of sodium nitrate and dipotassium-ortho-phosphate. The culture condition was maintained at 25 C under continuous uorescent light with light intensity of approximately 100 µmol m −2 s −1 , and the asks were hand shaken 2 to 3 times a day.
The Chlorophyll a content of Chlorella sp. T4 was measured as described previously [20]. The physiological and photosynthesis e ciency of the microalgal cells were studied as described previously [21]. The maximum quantum e ciency of Photosystem II (PS II) was calculated using the equation: F V /F m = (F m -F 0 )/F m as previously described [21], Where F m , F o, and Fv represents the maximum, minimum and variable uorescence, respectively.

Measurement of cell growth, biomass concentration, lipid yield and fatty acid content
Cell growth determination, biomass concentration measurement, and algal lipids extraction and weight determination were carried out as described previously [15]. The harvesting was done by centrifugation at 5000 rpm for 10 min. The fatty acid content were quanti ed as described previously [15] while the biodiesel properties were estimated using the web version of the Biodiesel Analyzer 2.2 [22].

Gene expression analysis
The expression levels of ve key fatty acid biosynthetic genes (rbcL, accD, KAS-1, ω-6 FAD and ω-3 FAD) were determined in samples collected at the early log phase (day 7), late log phase (day 14) and stationary phase (day 21) of growth in the different nutritional growth conditions. The total RNA was extracted from ≈100 mg of algal cells using GeneJet RNA puri cation kit (Thermo Fisher Scienti c, MA, USA) followed by synthesis of rst-strand cDNA using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scienti c, MA, USA) according to the manufacturer's instruction. The level of gene expression was monitored by Real-time quantitative PCR performed with Universal SYBR Green Supermix (Bio-Rad, CA, USA) in Hard-Shell High-Pro le 96-Well Semi-Skirted PCR Plates (Bio-Rad, CA, USA) using 50 ng cDNA as the template and primer pairs listed in Table 1. Table 1 Primers used in the real time RT-PCR for quantifying the biosynthetic genes.

Gene
Sequence (

Statistical analyses of experimental results
The data were analysed by one-way ANOVA at 95% con dence limit (α = 0.05). All statistical tests were performed using SPSS (v. 20, IBM). Unless otherwise stated, p < 0.05 denotes a statistically signi cant difference. The values were expressed as the mean ± standard deviation.

Cell growth and biomass accumulation
The effects of varying nutrient concentrations on the growth of Chlorella sp. T4 were investigated to ascertain suitable condition for biomass yield and high lipid productivity. Cultivation of microalgae under nutrient limiting condition has been reported to decrease the overall algal biomass, while inducing synthesis of neutral lipid suitable for biodiesel production [12]. In this study, nutrient stress conditions produced adverse effects on the proliferation of Chlorella sp. T4 cells (Fig. 1). As shown in Table 2, low speci c growth rates 0.055 ± 0.004 h −1 was observed when Chlorella sp. T4 was cultivated under Nlimiting medium, with short generation time of 0.079 ± 0.005 day −1 compared to the control. High speci c growth rate of 0.079 ± 0.004 h −1 was observed under N-replete medium which was not signi cantly higher than that the growth rate in the control medium. This is also re ected by similar growth pattern of Chlorella sp. T4 obtained in N-replete medium and control medium (Fig. 1a). It proves the importance of nitrogen as a macro nutrient required for protein synthesis and cell division in microalgae [11]. The observed growth patterns under N and P-limiting medium ( Fig. 1) are consistent with those reported for other Chlorella strains [17,14,25].  The microalgae Chlorella sp. T4 showed tolerance to high N concentration as demonstrated by the growth curve similar to that obtained in the control medium. Similarly, higher speci c growth rate was observed in medium with higher concentration of phosphorus. The speci c growth rate (0.050 ± 0.011 h −1 ) observed under P-limiting condition is about two-fold less compared to the value obtained in the Preplete medium (0.098 ± 0.014 h −1 ). This was also corroborated by the growth patterns of the strain under P-limiting condition (Fig. 1b). P-replete medium produced higher generation time of 0.147 ± 0.020 day −1 , which is 1.35-and 2.04-fold higher than the generation times obtained in the control and P-limiting medium. Phosphorus is a constituent element of ATP, essential for photophosphorylation which has signi cant relevance to the cell growth and metabolism of microalgae. Photosynthetic microalgae require large amounts of proteins (mainly RuBisCO) which is synthesized by phosphorus-rich ribosome [26].
For all the experiments, biomass yield and productivity together with lipid content and productivity were calculated after 21 days cultivation period. Nitrogen replete medium (N-2.25) produced the highest biomass yield of 0.82 ± 0.06 g L −1 which is 41.4% signi cantly (p 0.05) higher than the biomass yield in the nitrogen de ciency (N-0.75) medium but not signi cantly different from that obtained in the control medium (Table 3). Similarly, 23.4% signi cantly (p 0.05) higher biomass yield was obtained in P-replete (P-0.06) medium compared to the P-de ciency (P-0.02) medium. This is further correlated by the high biomass productivity 38.95 ± 0.84 mg L −1 d −1 obtained under N-replete medium (38.95 ± 0.84 mg L −1 d −1 ) and P-replete medium (37.52 ± 0.53 mg L −1 d −1 ) due to high nutrient availability to utilize for cell division (Table 3). Reduction in nutrients concentration in media has been shown to slow down the metabolic activity and cell division in most microalgae, while triggering lipid accumulation [27]. It is therefore not surprising that a signi cantly 1.6-fold and 1.2-fold increases in lipid productivity and lipid content were obtained in nitrogen limiting and phosphorus limiting medium, respectively, relative to the nutrient replete medium. High biomass productivity by this microalgae strain under both nutrient rich and nutrient stressed conditions is very promising, since biomass productivity is one of the major traits that makes microalgae attractive feedstock for biofuel applications over plant-based feedstocks [28]. Similarly, [29] found that an increase in N concentration resulted high biomass yield of 1.56 and 1.78 g L −1 for Chlorella sorokiniana (PCH02) and Chlorella vulgaris (PCH05), respectively. The observed overall increase in biomass yield and biomass productivity in N and P rich medium of this strain may be due to luxurious uptake of phosphorus which gets deposited in the cell as polyphosphate involved in metabolic pathway and storage for further use during phosphorus starvation/ limitation [30]. Similarly, [25] reported an increase in biomass concentration of Chlorella minutissima MCC as the phosphate concentration increase from 0 to 3 mM.

Chlorophyll content and physiological response of Chlorella sp. T4 under different nutrient conditions
Chloroplast is an important unit component for most photosynthetic plants and algae. Hence, the chlorophyll content and the viability of the photosynthetic process are critical physiological indicator for monitoring microalgae adaptability to different culture conditions [14]. The chlorophyll a content was measured during the cultivation period as an indicator of the physiological response of Chlorella sp. T4 in different N and P conditions. An increase in chlorophyll a content was observed as the concentration of N and P increased in media. Highest chlorophyll a content (in mg/g dcw) of 27.11 ± 0.01 (Fig. 2a) and 26.50 ± 0.67 (Fig. 2b) was observed after 21 days in N and P-replete medium, respectively. A signi cantly (p 0.05) decrease in chlorophyll a content by 1.2-fold and 1.4-fold under N and P-de cient conditions compare to the control, respectively, were recorded. It suggests that the decrease of N and P concentration in the medium results in lower cell chlorophyll accumulation due to scarcity of intracellular nitrogen and phosphorus pool to synthesize chlorophyll for further cell reproduction. Nitrogen and phosphorus are most important elements contributing to the growth of microalgae cell, it limitation signi cantly changes the biosynthesis of cell pigment [31]. The results clearly show the in uence of nutrient limitation on the growth physiology of Chlorella sp. T4 as the growth rate and biomass yield also decreased under these conditions.
The widely used uorescence parameter Fv/Fm, an index re ecting irradiance acclimation status [32] was investigated under nutrient de cient and replete medium. It represents the measure of photosystem II (PSII) quantum yield and could be used to evaluate the photo induced damage to protein complex [33].
The positive in uence of N and P treatment conditions was observed on the growth of Chlorella sp. T4 (Fig. 2c,

Analysis of lipid content and composition
Studies have shown that cultivation of microalgae under nutrients de ciency conditions stimulates lipid biosynthesis in many microalgae species [3,8]. In this study, Chlorella sp. T4 showed a signi cant (p 0.05) increase in total lipid yield under N and P-limiting conditions, accounting for 31.07 ± 0.53% and 28.33 ± 1.35% of dry cell weight, respectively (Table 3). [35], cultivated C. zo ngiensis under N and Pde cient medium and reported higher lipid contents as compared to the nutrient su cient medium which is similar to the present study. [3] also observed high lipid accumulation by Chlorella sorokiniana under N and P-de cient medium compare to the control medium. Contrary, [12] cultivated Chlorella sp. in medium containing different N concentration and observed an increase in lipid accumulation under N-replete medium compare to N-de cient conditions.
Lipid productivity is one of particular importance microalgal lipid production process as it considers both lipid content and biomass production rate. High lipid productivity of 15.54 ± 0.7 mg L −1 d −1 was obtained under N-limiting condition which was 1.37-fold higher than P-limiting condition after 21 days (Table 3). Nonetheless, nutrient limiting conditions repressed the growth of Chlorella sp. T4 and the overall productivity caused by nutrient de ciency was not offset by biomass loss. Similarly, [14] reported high lipid productivity of 47.05 mg L −1 d −1 under N-de ciency in Chlorella pyrenoidosa after 5 days of cultivation. Also, [36] investigated the effects of phosphorus on lipid accumulation of Chlorella vulgaris and reported a lipid productivity of 19.40 mg L −1 d −1 in phosphorus de cient medium. Low lipid productivity was observed under N-replete medium (10 ± 0.15 mg L −1 d −1 ) which was 2.32-fold lower than the control medium. These ndings clearly show that high lipid productivity yield can be obtained by cultivating microalgae under nutrient de ciency conditions that has just enough nutrients to support the growth.
Microalgae biomass contains signi cant quantities of lipids in the form of triacylglycerol, which can be converted to biodiesel via transesteri cation process. This has attracted huge commercial interest of using microalgae as feedstock for biodiesel production [28]. The lipid composition of Chlorella sp. T4 varied according to the nutrient concentration of the growth medium (Table 4). Previous studies have also shown that the concentration of nitrogen and phosphorus in microalgae culture alter total fatty acid content and composition [17,37]. Palmitic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2) constitute the major fatty acids in algal oil. Fatty acids of this chain length are reported to be suitable for high quality biodiesel production [1]. Further analysis reveals that saturated fatty acid (SFA) ranged from  N-0.75, N-limiting condition; N-2.25, N-replete medium; P-0.02, P-limiting condition and P-0.06, Preplete medium; Control, BG-11 containing N-1.5 g L −1 + P-0.04 g L −1 . Different letters depict signi cance difference among the group according to one-way ANOVA at p < 0.05. Mean value shown is the average of three replicates ±SD.
There was high level of saturated fatty acid observed under P-replete (49.9%) medium, which is 1.3-fold higher than the control medium (Table 4). High level oleic acid (37.1 ± 0.1%) was obtained in the control medium, but not signi cantly (p 0.05) different from the level obtained in both the N and P-limiting medium. High content of oleic acid is bene ciary for excellent oxidative stability, increases biodiesel's ow properties and reduces it solidi cation temperature [38,39]. Furthermore, high content of PUFAs was found in P-limiting condition which could cause decline of cetane number and oxidation stability, making biodiesel prone to oxidation-dependent degradation [38,40]. This nding was well with the results reported by [17] who reported that high accumulation of PUFAs was obtained under P-limiting condition.
The quality of microalgae biodiesel is measured by the important thermophysical properties of biodiesel and comparing those with the international standards such as ASTM D675 or EN14214 (Table 5). Previous studies have demonstrated that fatty acid pro le signi cantly affected the quality of biodiesel [22,1]. The oxidative stability of the biodiesel produced in this study ranged between 5.75 to 7.20 h which is favourable for biodiesel production due to saturated fatty acid [41]. This microalgae strain showed low cold lter plugging properties (-2.75 ºC) under N-limiting condition which is preferable for biodiesel production. This was caused by a good balance between the saturated fatty acid and monounsaturated fatty acid observed under the N-limiting condition.  High saturated fatty acid content may reduce the cold lter plugging point properties of biodiesel because saturated fatty acid has higher melting points than unsaturated fatty acid [42]. Furthermore, kinematic viscosity (mm 2 s −1 ) produced by Chlorella sp. T4 under all N and P conditions was outside the range recommended by ASTM D675 and EN14214. This property may result into biodiesel produced with high viscosity affecting the fuel atomization and lead to deposits forming inside the engine, due to high PUFAs contain by microalgae compare to the other seed oils. Linoleic acid was above 12 recommended by EN14214 for all the conditions, an indication of poor oxidative stability with good cold ow properties [28]. In this study, the best biodiesel was produced under N-limiting condition, with high ignition quality, good oxidative stability, cetane number value and saponi cation value (Table 5).

Effect of culture conditions on the expression of rbcL and accD genes of Chlorella sp. T4
During photosynthesis, the RuBisCo enzyme is involved in carbon xation process. A large subunit of this enzyme encoded by gene rbcL which harbour binding site [43,44]. The present study evaluates the effect of nutrient conditions on the expression levels of some functional and fatty acids biosynthetic genes from Chlorella sp. T4. A signi cant (p 0.05) decrease of 2.09-fold in the rbcL gene expression was observed under N-limiting condition after 21 days compare to the control (Fig. 3a). There was no signi cant difference in the expression level of rbcL under N-replete compared to the control medium (Fig. 3a). Similarly, the rbcL gene was signi cantly (p 0.05) decreased by 1.59-fold under P-limiting condition after 21 days compare to the control (Fig. 3b). Under nutrient stress, the cell protein synthesis and photosynthetic rates is affected as chlorophyll a is utilized as an intracellular nitrogen to support the growth of microalgae [45]. [14] cultivated Chlorella pyrenoidosa under N and P-de cient conditions and reported high expression of rbcL gene in the nutrient condition which was two to ve times higher than Nde cient condition. [3] also reported 78% and 56% down regulation of rbcL gene in N and P stress conditions on Chlorella sorokiniana, respectively. The decrease in the expression of rbcL gene was also translated by low speci c growth rate ( Table 2) and decrease of maximum quantum e ciency of PSII (Fig. 2) under N-limiting condition in this study. The expression of rbcL gene was signi cantly (p 0.05) increased by 1.12-fold under P-limiting condition compared to the control medium just after 7-day incubation periods. (Fig. 3b). Microalgae utilizes phosphorus for the transfer and signal transduction during photosynthesis [46]. Microalgae under nutrient su cient medium tend to require more xed carbon cell construction, which then demand for more RuBisCO to sequester the CO 2 in the air.
Acetyl-CoA carboxylase (ACCase) is regarded as rate-limiting enzyme for fatty acid synthesis and it has been overexpressed in different organism to enhance lipid production [6]. A study by [14] suggested a strong involvement of accD in triggering lipid accumulation by the cell under nutrient de cient conditions. The present study evaluated the expression of heteromeric ACCase unit (accD gene) as a function of different N and P concentrations on lipid synthesis. A signi cant increase in the expression of accD gene was observed under nutrient limiting conditions during the cultivation period as compared to the cells grown in standard BG-11 medium (Fig. 3c & d). In N-limiting condition, 3.11-fold increase of accD gene expression was observed after 21 days cultivation compared to the control (Fig. 3c). Likewise, a signi cant (p 0.05) 1.89-fold increase in the expression accD gene by was observed under P-limiting condition after 21 days of cultivation compared to the control (Fig. 3d). Usually, lower photosynthetic rates cause NADH accumulation inhibiting enzyme citrate synthase so that the acetyl-CoA is blocked from entering TCA cycle. By increasing the acetyl-CoA concentration, ACCase is activated resulting in the enhancement of lipid content in microalgae [6]. This was evidently shown by an increase lipid yield by Chlorella sp. T4 under N and P-limiting conditions compare to the control medium (Table 3). During nutrient starvation, cell tends to synthesis lipids as a protective mechanism against stressful condition [47].
The expression of accD gene under N-replete medium was signi cantly lower compared to the control and N-de cient medium, with 1.3-fold and 2.6-fold increase obtained, respectively at day 21 (Fig. 3c).
Similarly, the expression of accD gene under P-replete medium was signi cantly lower by 2.95-fold compare to the control at day 21 (Fig. 3d). [3], cultivated Chlorella sorokiniana under N and P-limiting conditions along with metal stress. They recorded a 3.24-fold and 2.93-fold increase in the expression of accD gene at the late log phase compared to the control medium (BG-11). Also, a signi cant correlation was found between the expression of accD gene, growth rate, photosynthetic e ciency, and lipid accumulation. Based on the results obtained in the present study, the expression of accD was observed to be higher under nutrient limiting medium. This was attributed by higher amount of lipid content under nutrient limiting medium despite lower biomass yield compared to N-replete medium.
3.5. Effect of culture conditions on the expression of KAS-1, ω-6 and ω-3 desaturase gene of Chlorella sp. T4 Another key gene in the process for fatty acid biosynthesis is KAS-1 which is required for the addition of malonyl-CoA to elongate a 4-carbon fatty acid to 6-, 12-and 16 carbon fatty acid chains [18]. There was no signi cant difference in the expression level of KAS-1 gene under N-de ciency and control medium after 21 days as observed in this study (Fig. 4a). The expression of KAS-1 gene was signi cantly (p 0.05) increased under N and P-replete medium by 1.12-fold (Fig. 4a) and 1.19-fold (Fig. 4d) after 21 days compare to the control, respectively. Usually, microalgae under normal growth condition consume ATP and NADPH produced by the cell though photosynthesis resulting in the formation of ADP and NADP + that are being available again as acceptor molecules in photosynthesis [48]. This was translated by high biomass ( Table 3) and abundance of saturated fatty acid (Table 4) observed in N and P-replete medium which can be attributed to the high expression level of KAS-1 gene under these conditions.
Aziz et al. [17] cultivated Chlorella strain KS-MAS under different P concentration and observed high expression of KAS-1 gene under P-replete condition, which is about 3.7 and 4.3-fold higher than the control. They found a strong correlation between the expression of KAS-1 and saturated fatty acid and biomass yield. The KAS-1 gene is known to catalyze the production of 18-carbon fatty acid from 16carbon fatty acid in which palmitic and steric acid are the nal product of the fatty acid synthesis [49]. Omega-6 desaturase encoded by gene ω-6 FAD catalyzes the conversion of oleic acid to linoleic acid. The expression level of ω-6 FAD by Chlorella sp. T4 was affected by nutrient conditions. The expression of ω-6 FAD was signi cantly (p 0.05) increased under N-limiting condition by 2.09-fold at day 21 compared to the control (Fig. 4b). The increase in the expression of ω-6 FAD under N-limiting condition was corroborated by high level of linoleic acid ascertain under N-limiting condition. Microalgae requires su cient ω-6 FAD gene expression to convert oleic acid substrate to linoleic acid [51]. The expression of ω-6 FAD gene was signi cantly decreased in N-replete medium by 1.83-fold compare to the control medium (Fig. 4b). [52] reported high expression of ω-6 FAD gene by Nannochloropsis oceanica under Nstarvation condition which led to an increase in linoleic acid content.
The expression of ω-6 FAD was signi cantly increased under P-limiting condition by 1.97-fold after 21 days compare to the control (Fig. 4e). The increase in the expression of ω-6 FAD under P-limiting condition was corroborated by high level of oleic acid ascertain under N and P-limiting condition ( Table   4). Omega-6 desaturase is activated by the availability of oleic acid and α-linoleic acid. This suggests that the function of desaturase enzyme was satisfaction to the demand of membrane phospholipids for synthesis of PUFAs. The present study demonstrated that nutrient limiting conditions had a signi cant impact on the expression of ω-6 FAD and monounsaturated fatty acids.
Omega-3 desaturase encoded by gene ω-3 FAD plays important role in the conversion of linoleic acid to form trienoic fatty acid known as α-linoleic acid [53]. The expression of ω-3 FAD gene was signi cantly (p 0.05) increased by 1.93-fold and 1.65-fold under N and P-limiting condition after 21 days compare to the control, respectively (Fig. 5c, f). The expression levels of ω-3 FAD gene is strongly associated with the increase in α-linoleic acid level [51], which was corroborated by high levels of α-linoleic acids content that was ascertain under N and P-limiting conditions by Chlorella sp. T4 (Table 4).
[54], reported overexpression of ω-3 FAD gene by Chlorella vulgaris in transgenic tobacco plant which resulted to an increase in α-linoleic acids content. The expression of ω-3 FAD gene was signi cantly (p 0.05) decreased under N and P-replete medium by 2-fold and 1.89-fold after 21 days compare to the control, respectively (Fig. 5c, f). Nevertheless, the accumulation of α-linoleic acid by Chlorella sp. T4 was relatively low to compare to other fatty acid. Omega-3 desaturase gene have been successfully overexpressed to increase the α-linoleic acids content [52,51]. Polyunsaturated fatty acid are major constituents of biological membrane which plays important role in maintaining the membrane uidity and are essential for cell growth at low temperatures [53].

Conclusion
The cultivation of Chlorella so. T4 in nutrient replete medium has resulted in increase in the cell growth rate which was attributed by high chlorophyll content and quantum e ciency of photosystem II (Fv/Fm) value. The biomass was signi cantly decreased under nutrient-stress condition, as corroborated by signi cantly decrease in the expression of rbcL gene. The correlation between the upregulation of accD gene and enhanced lipid productivity by N and P limitation was observed indicating a clear impact of nutrient stress in Chlorella sp. T4. The level of KAS-1 gene was upregulated under nutrient replete medium, translated by high level of saturated fatty acid under non-nutrient stress conditions.
Furthermore, an increase in the expression level of ω-6 FAD and ω-3 FAD genes under N and P-limiting medium was observed which corresponded to high levels of monounsaturated and polyunsaturated fatty acid. This provides a clue for future prospective metabolic engineering to make microalgal biodiesel economically viable. FAMEs produced under nutrient limiting condition were suitable for production of high-quality biodiesel with better oxidative stability and cold ow properties. Future research may focus on the overexpression of these key biosynthetic genes through metabolic engineering for higher yield of neutral lipid with good biodiesel properties.